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Stephen Adeniyi Adefegha1,*, Olorunfemi Raphael Molehin2
1Functional Foods and Nutraceuticals Unit, Department of Biochemistry, Federal University of Technology, Akure, Nigeria P.M.B., 704, Akure 340001, Nigeria

2Department of Biochemistry, Faculty of Science, Ekiti State University Ado-Ekiti, P.M.B.5363 Ado-Ekiti, Nigeria

*Corresponding Author address: Functional Foods and Nutraceuticals Unit, Department of Biochemistry, Federal University of Technology, Akure, Nigeria P.M.B., 704, Akure 340001, Nigeria. E-mail � saadefegha@futa.edu.ng Tel: +2348034350812













Abstract
Brimstone (Morinda lucida) root is one of the widely used herbs in folkore medicine for the management of diabetes while acarbose is a well-known antidiabetic drug. This study sought to characterize the phenolic constituents of brimstone (Morinda lucida) root, assess its interaction with key enzymes (�-amylase and �-glucosidase) relevant to type-2 diabetes and evaluate its effect on acarbose in vitro. One milligram per millilitre of aqueous extract of brimstone root and acarbose were separately prepared. In addition, both the brimstone and acarbose solution (50:50 v/v) were thoroughly mixed until homogeneity was attained. The phenolic phytoconstituents of brimstone root were subsequently determined. Finally, the effects of brimstone root extract, acarbose solution and a mixture of acarbose and brimstone extract on a-amylase and � -glucosidase activities were assessed in vitro. Brimstone root extract was found to be rich in phenolic acids (p-coumaric acid, gallic acid, caffeic acid and chlorogenic acid) and flavonoid (apigenin). Acarbose had significantly (p < 0.05) higher � -amylase (IC50= 0.02 mg/ml) and �-glucosidase (IC50= 0.01 mg/ml) activities when compared to brimstone root extract [� amylase (IC50=0.62 mg/ml); �-glucosidase (IC50= 0.33 mg/ml)]. Furthermore, the combination of acarbose and brimstone root extract showed synergistic effects on �-amylase and �  glucosidase inhibition (in vitro). This study may thus suggest that brimstone root represent a source of phenolic phytochemicals for the management of non-insulin dependent diabetes mellitus.

Keywords: Brimstone, Acarbose, �-Amylase, �-Glucosidase, Type-2 diabetes









Introduction
Diabetes mellitus is described as a chronic metabolic disease characterized by elevated blood glucose (hyperglycaemia), which is a result of defects in insulin resistance, secretion of insulin and dysfunctionalties of beta cells [1, 2]. Among the diagnosed cases of diabetes mellitus, type 2 diabetes (T2D) accounts for the most prevalent cases with approximately 90-95% [3]. Diabetes has been attributed to several risk factors involving lifestyle, such as smoking, obesity, poor diet and physical inactivity [4]. �-Amylase and � -glucosidase are major enzymes involved in starch hydrolysis and are implicated in the onset of type 2 diabetes (T2D) [5-8]. Pancreatic �-amylase is an that catalyzes the breakdown of starch to a mixture of oligosaccharides and disaccharides, while �-glucosidase found in the intestines breaks down disaccharides and oligosaccharides into glucose which, upon absorption, moves into the bloodstream [8-9]. The breakdown of these complex carbohydrates is very swift and may result in an elevation of blood glucose level after a meal [10]. The oral administration of antidiabetic drugs from synthetic sources like acarbose and miglitol elicit their therapeutic effects by inhibiting these enzymes, but their administration is also accompanied with attendant side effects and a failure to alleviate diabetic complications [11-12]. In recent times, the burst of interest in the use of medicinal plants in the form of traditional medicine has greatly increased all over the world, most especially in countries of Africa [13-14].
In Nigeria, a large portion of the populace relies on herbal medicine because it is cheap, effective and it is readily accessible [15-16]. Several medicinal plants commonly used for management/treatment of various types of ailments exists out of which is Morinda lucida Benth (Morinda lucida) (L.) which belongs to the family Rubiaceae. It is found in tropical West Africa rainforest and its common name is Brimstone tree [17]. The tree is around 15m tall with scaly grey bark, short crooked branches and shining foliage. The plant parts (leave, stem, root and bark) are used in folklore medicine to treat some ailments and it has been reported to possess strong trypanocidal and aortic vasorelaxant activities [16], anticancer [17], hepatoprotective [18], antispermatogenic [19], hypoglycemia and antidiabetic [20] properties. Adeneye and Agbaje [17] has previously reported the antihyperglyceamic effect of Morinda lucida extract in alloxan-induced diabetic rats. However, there is dearth of information on the possible mechanism of actions of the plant in exhibting its antidiabetic effects. Consequently, we evaluated and compared the inhibitory effect of Brimstone root extract and acarbose (a known antidiabetic drug) on �-amylase and �-glucosidase activities in vitro, with the view to explain the possible mechanisms that may be involved in the combinatorial usage of traditional medicine and synthetic drug.
Materials and methods
Sample collection
Brimstone (Morinda lucida) roots were purchased from local traditional healers at the Erekasan Market in Akure Metropolis of Ondo State, Nigeria. The plant was authenticated at the Department Biology, Federal University of Technology, Akure (FUTA) Nigeria by A.A. Shorungbe and given the voucher number BIO/FUTA/035.
Reagents and chemicals 
Methanol, gallic acid, phosphoric acid, caffeic acid, p-coumaric acid and chlorogenic acid were sourced from Merck (Darmstadt, Germany). Apigenin was obtained from Sigma Chemical Co. (St. Louis, MO, USA). Except otherwise stated, chemicals and reagents used in this experiment were of analytical grade and glass-distilled water was used throughout the study. The absorbance was read by UV/visible spectrophotometer (Jenway 6305 model, Bibby Scientific) while the centrifugation was carried out with the aid of Kenxin refrigerated centrifuge (KX3400C, KENXIN Intl. Co., Hong Kong).
Staffordshine, United Kingdom) was
Aqueous extraction of Brimstone roots and acarbose
Morinda lucida roots was thoroughly washed under running distilled water, air dried, ground into powder and kept dry in an air-tight container prior to extraction as described by Adefegha and Oboh [21]. The sample (5g) was weighed, soaked in 100 ml of distilled water and left for 24h. The mixture was filtered after 24h and the filtrate kept in a refrigerator below 40C. The frozen extract solution was recovered as dried extract using a freeze drier. The reconstitution of the dried extract was done in distilled water (1mg/ml) and used for subsequent analysis. Likewise, a stock concentration of 1 mg/ml acarbose was prepared and later diluted for subsequent use.
Determination of total phenol content
The total phenol content was determined on the extracts using the method reported by Singleton et al. [22] Appropriate dilutions of the extracts were oxidized with 2.5mL of 10% Folin Ciocalteau s reagent (v/v) and neutralized by 2.0mL of 7.5% sodium carbonate. The reaction mixture was incubated for 40 min at 45 �%C and the absorbance was measured at 765nm in the spectrophotometer. The total phenol content was subsequently calculated using Gallic acid as standard.
Determination of total flavonoid content
The total flavonoid content of both extracts was determined using a slightly modified method reported by Meda et al.[23]. Briefly, 0.5mL of appropriately diluted sample was mixed with 0.5mL methanol, 50 �L of 10% AlCl3, 50 �L of 1mol L"1 potassium acetate and 1.4mL water, and allowed to incubate at room temperature for 30 min. Thereafter, the absorbance of the reaction mixture was subsequently measured at 415 nm. The total flavonoid was calculated using quercetin as standard.
Quantification of phenolic compounds by High performance liquid chromatography couple with diode array detector (HPLC-DAD)
The phenolic profile of brimstone extract was determined by a reverse phase chromatography analysis under gradient conditions using the method of Adefegha et al.[24] with slight modifications. The peaks of the chromatogram were confirmed by comparing their retention time with those of reference standards and by DAD spectra (200 to 600 nm). The limit of detection (LOD) and limit of quantification (LOQ) were calculated based on the standard deviation of the responses and the slope using three independent analytical curves, as defined by Cruz et al. [25] LOD and LOQ were calculated as 3.3 and 10 �/S, respectively, where � is the standard deviation of the response and S is the slope of the calibration curve. The HPLC analyses were performed by using the free software R version 3.1.1 [26]

�-Amylase inhibition assay
The extract of Brimstone (500�L) and 500�l of 0.02M sodium phosphate buffer (pH 6.9 with 0.006 M NaCl) containing Hog pancreatic �-amylase (EC 3.2.1.1) (0.5mg/mL) were incubated at 25�C for 10minutes. Then, 500�l of 1% starch solution in 0.02M sodium phosphate buffer (pH 6.9 with 0.006 M NaCl) was added to each tube. The reaction mixtures was incubated at 25�C for 10minutes and stopped with 1.0mL of dinitrosalicylic acid colour reagent. Thereafter, the mixture was incubated in a boiling water bath for 5minutes, and cooled to room temperature. 10mL of distilled water was added to the reaction mixture was and the absorbance measured at 540 nm [27]
2.8. �-Glucosidase inhibition assay
(50�l) extract of Brimstone and 100�l of �-glucosidase solution (1.0 U/mL) in 0.1M phosphate buffer (pH 6.9) was incubated at 25�C for 10min. Then, 50�l of 5mM p-nitrophenyl-�-D-glucopyranoside solution in 0.1M phosphate buffer (pH 6.9) was added. The mixtures were incubated at 25�C for 5min, before reading the absorbance at 405nm in the spectrophotometer. The �-glucosidase inhibitory activity was expressed as percentage inhibition [28].
2.9. Statistical analysis
Results of triplicate experiments were pooled and expressed as mean � standard deviation (SD). The IC50 (extract concentration causing 50% enzyme inhibitory/antioxidant) was calculated by a non-linear regression using Graph Pad Prism version 6.0 software. Differences between groups of HPLC determinations were assessed by analysis of variance and Tukey's test was performed. The level of significance was defined as p < 0.05. 

Results 
Table 1 reveals the total phenolic contents of Brimstone root. The result showed the total phenolic content of 5.78�0.12 mgGAE/g and total flavonoid content of 2.19�0.14 mgQUE/g. Furthermore, the HPLC-DAD analysis was performed on the Brimstone root to identify and quantify the presence of phenolic acids and flavonoids. The results (table 3 and figure 5) indicates that the plant extract contains p-Coumaric acid (1.06 � 0.04 mg/g) and apigenin (1.18 � 0.01 mg/g) and chlorogenic acid (0.73 � 0.01 mg/g) as the predominant phenolic compound. Other phenolic compounds found in Brimstone roots include, gallic acid (0.29 � 0.03 mg/g), caffeic acid (0.35 � 0.02 mg/g).

�- amylase and �-glucosidase inhibitory activities
The �-amylase inhibitory properties of acarbose and brimstone extract were assessed and presented in Figure 1. The result revealed that both acarbose and the root extract inhibited �-amylase activity in a concentration-dependent manner. Acarbose (IC50= 13.75 �g/ml) had significantly (p<0.05) higher �-amylase inhibitory effect than brimstone extract (IC50 = 0.62 mg/ml) as shown in Table 2.  However, the in vitro effect of combination of Brimstone root extract with acarbose was investigated on their �-amylase inhibitory activities (Figure 2). The combination of the synthetic drug (acarbose) and traditional plant (brimstone root) resulted into significant (p<0.05) increase in the �-amylase inhibitory activity when compared with the plant extract only and synthetic drug (acarbose) alone. Likewise, the �-glucosidase inhibitory abilities of the plant, acarbose and their combination are presented in Figures 3 & 4 and the IC50 in Table 2. The result revealed that acarbose (IC50= 8.25 �g/ml) had significantly (p<0.05) higher �-glucosidase inhibitory activity than Brimstone (IC50 = 0.33 mg/ml). Also, the combination of Brimstone and acarbose (50:50 v/v) also caused a significant (p<0.05) increase in the �-glucosidase inhibitory activity when compared with Brimstone only and acarbose alone.

Discussion
Polyphenols are one of the most abundant antioxidants in plants and they are widely distributed across several food products such as herbs, vegetables, cereals, olive, dry legumes, chocolate and beverages [29-31]. In this study, the values obtained for the total phenol and flavonoid contents of Brimstone root extract are higher than that reported for some Nigerian spices 21 but lower than that reported by [32] on Embelia philippinensis as well as the recent study from our group on African birch leaves[12].
Inhibition of �-amylase and �-glucosidase activities by Brimstone extract may suggest the scientific basis for its folkloric use in the management/ treatment of diabetes and could be of great value in the development of a modern therapeutic intervention in the management of type-2 diabetes (T2D) [10, 33-34]. Furthermore, p-Coumaric acid apigenin and chlorogenic acids were present in abundance when compared with other phenolics found in Brimstone root. Recent reports have shown that p-Coumaric, apigenin and chlorogenic acids are good inhibitors of �-amylase and �-glucosidase activities. [10, 34-35]. Therefore, we suggest that the inhibition of �- amylase and �-glucosidase activities by plants and plant foods may be linked to its phenolic constituents [29, 36-37]. This agrees with some studies where inhibition of key enzymes (�-amylase and �-glucosidase) relevant to T2D by plants have been speculated to serve as therapeutic targets in the management of this disease, and some vital bioactive compounds including polyphenols may possess interesting structure-function benefits and promising potentials. [10, 12, 38-39]. The increased inhibition of �-amylase and �-glucosidase activities by acarbose and Brimstone extract when combined (50:50) resulted into synergistic action/effect (Figure 2 and 4). This property may confer advantage over synthetic drugs in the management of postprandial blood glucose, such as Acarbose, which strongly inhibit � -amylase. Stronger inhibition of �-glucosidase activity and mild inhibition of � -amylase activity of the brimstone root extracts could address the major drawback of currently used �- glucosidase and �-amylase inhibitory drugs with side effects such as abdominal distention, flatulence, meteorism and possibly diarrhea [5,9]. It has been suggested that such adverse effects might be as a result of excessive pancreatic �-amylase inhibition leading to the abnormal bacterial fermentation of undigested saccharides in the colon, Findings from this study correlates with earlier study by Boath et al. [38] who affirmed that synergies between acarbose and berry extracts in diabetes treatment/management may enhance hypoglycemic effect and inhibition of �-glucosidase activity. Recent report from our group has shown that the combination of acarbose and African birch extract had an additive effect on �-amylase inhibition, while a resultant synergistic action was observed against �-glucosidase inhibition [12].The clinical relevance of herb-food interaction may be of beneficial effect by increasing drug efficacy or diminishing potential sideDt������
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Conclusion
This study reports the phenolics of brimstone root, the synergistic interaction of Brimstone and acarbose in inhibiting �-amylase and �-glucosidase activities respectively. However, these effects could be attributed to some phenolic components of Brimstone root.
Conflict of Interest:  
The authors declare no conflict of interest.
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